In this research project, we propose to study the molecular mechanisms by which RE1 silencing transcription factor (REST) regulates cardiomyocyte cell cycle using synergistic approaches of mouse genetics, molecular and systems biology. Our ultimate goal is to identify potential therapeutic targets to combat heart disease by promoting cardiomyocyte proliferation to repair the injured adult heart. The adult heart has a limited repairing capacity, because most adult cardiomyocytes are non-dividing cells. For adult heart regeneration by pre- existing cardiomyocytes, we need to understand how cardiomyocyte proliferation is controlled, what population of cardiomyocytes maintains proliferation potential, whether developmental mechanisms of cardiomyocyte proliferation are re-activated under diseased conditions, and if they can be further enforced. Here we plan to address these questions by investigating REST functions in the mouse heart, because our recent studies have identified REST as a new intrinsic regulator of cardiomyocyte proliferation required for embryonic heart development and neonatal heart regeneration. REST represses the cell cycle inhibitor p21 to maintain cardiomyocyte cell cycling. REST is also required for the expression of several key cell cycle activators. Based on these findings, we hypothesize that upregulation of REST may improve the renewal of the injured heart by modulating the expression of cell cycle genes and regulators to promote cardiomyocyte proliferation. We will test this hypothesis in two aims. Aim 1 will determine whether forced REST expression in failing hearts promotes cardiomyocyte cell cycle and improves the disease outcome. We will specifically inactivate REST in the cardiomyocytes, with or without the p21 inactivation, as well as forced REST expression, to determine whether REST is required for adult heart repair. Aim 2 will investigate how REST controls cardiomyocyte cell cycle in normal and failing hearts by studying and comparing the REST regulatory network in the mature (MYH6+) and immature (MYH7+) cardiomyocytes. We will also study the REST regulatory network by determining the REST recruitment of different chromatin co-factors for gene activation or repression. This will be achieved by using the cell-type specific, genome wide REST chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomics (RNA-seq) for MYH6+ versus MYH7+ cardiomyocytes from normal or injured hearts. The human relevance of mouse findings will be ascertained by the functional study of key REST- regulated genes in the proliferation of cardiomyocytes derived from the human inducible pluripotent stem cells (iPSC). By completing this project, we will have determined the REST role in promoting cardiomyocyte proliferation to improve the renewal and function of failing adult hearts. We will have also learned a genetic regulatory network by which REST regulates adult cardiomyocyte cell cycle and identified new targets for improving cardiomyocyte proliferation for heart regeneration. The new information will advance our understanding of cardiac biology and provide a new direction to treat heart failure.